Lessons Learned: Transfer of the High-Definition Circulating Tumor Cell Assay Platform to Development as a Commercialized Clinical Assay Platform.

Planning and transfer of a new technology platform developed in an academic setting to a start-up company for medical diagnostic product development may appear daunting and costly in terms of complexity, time, and resources. In this review we outline the key steps taken and lessons learned when a technology platform developed in an academic setting was transferred to a start-up company for medical diagnostic product development in the interest of elucidating development toolkits for academic groups and small start-up companies starting on the path to commercialization and regulatory approval.

Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor.

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain.

The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding to N-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity for N-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the N-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity for N-acetylgalactosamine is observed. The structure of this mutant reveals that the beta-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.

Structural basis of galactose recognition by C-type animal lectins.

The asialoglycoprotein receptors and many other C-type (Ca2+-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta-methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild-type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca2+ and form hydrogen bonds with amino acids that also serve as Ca2+ ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca2+ coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site.

Analysis of diffuse scattering from yeast initiator tRNA crystals.

Yeast initiator tRNA crystals exhibit strong X-ray diffuse scattering. This scattering can be used to extract information about lattice-coupled and intramolecular motions in the crystals. The amplitudes and correlation distances of these motions can be estimated by calculating the diffuse scattering and comparing the results with the observed scattering. Results indicate that both anisotropic, lattice-coupled motions as well as short-range correlated local disorder in the anticodon arm contribute to the overall disorder in the crystals. These types of motions can be correlated with aspects of tRNA function. This additional information complements the results from analysis of crystallographic data and provides a more detailed picture of the structure and dynamics of the molecule. The degree to which the methodology presented here can account for the observed diffuse scattering from tRNA represents a significant step forward in the ability to use this conventionally discarded information, and encourages the ultimate extension of these ideas to a wide variety of macromolecular systems.